T=1 capsid structures of Sesbania mosaic virus coat protein mutants: determinants of T=3 and T=1 capsid assembly

Abstract

Sesbania mosaic virus particles consist of 180 coat protein subunits of 29 kDa organized on a T=3 icosahedral lattice. N-terminal deletion mutants of coat protein that lack 36 (CP-NΔ36) and 65 (CP-NΔ65) residues from the N terminus, when expressed in Escherichia coli, produced similar T=1 capsids of approximate diameter 20 nm. In contrast to the wild-type particles, these contain only 60 copies of the truncated protein subunits (T=1). CP-NΔ65 lacks the "β-annulus" believed to be responsible for the error-free assembly of T=3 particles. Though the CP-NΔ36 mutant has the β-annulus segment, it does not form a T=3 capsid, presumably because it lacks an arginine-rich motif found close to the amino terminus. Both CP-NΔ36 and CP-NΔ65 T=1 capsids retain many key features of the T=3 quaternary structure. Calcium binding geometries at the coat protein interfaces in these two particles are also nearly identical. When the conserved aspartate residues that coordinate the calcium, D146 and D149 in the CP-NΔ65, were mutated to asparagine (CP-NΔ65-D146N-D149N), the subunits assembled into T=1 particles but failed to bind calcium ions. The structure of this mutant revealed particles that were slightly expanded. The analysis of the structures of these mutant capsids suggests that although calcium binding contributes substantially to the stability of T=1 particles, it is not mandatory for their assembly. In contrast, the presence of a large fraction of the amino-terminal arm including sequences that precede the β-annulus and the conserved D149 appear to be indispensable for the error-free assembly of T=3 particles.